This invention relates to a method of detecting mutations in nucleic acid sequences, and pertains more specifically to detection of mutations as small as single base changes.
Methods for detecting single base substitutions in nucleic acids provide powerful tools for the analysis of human genetic diseases (Orkin, et al., 1984 Ann. Rev. Genet. 18:131) and the establishment of human genetic linkage maps (Solomon, et al., 1979 The Lancet 1:923). Procedures currently available for detecting base substitutions rely on differences in restriction endonuclease cleavage sites (Flavell, et al., 1978 Cell 15:25), or on differences in the melting behavior of wild type and mutant DNA duplexes (Myers, et al., 1985 Nuc. Acid Res. 13:3111 and Myers, et al., 1985, Science 229:242). For example, some single base substitutions result in the loss or gain of a restriction endonuclease cleavage site, and can therefore be detected in Southern blotting experiments (Flavell et al., Idem). Another approach involves the use of synthetic oligodeoxyribonucleotides as differential hybridization probes (Wallace, et al., 1979 Nucl. Acids Res. 6:3543). In this method, a labelled synthetic oligonucleotide homologous to the mutant or wild type DNA is hybridized to blotted genomic DNA. Hybridization and washing conditions are then adjusted to allow the differential melting of the mismatched and perfectly paired duplexes. This method is useful for assaying the presence of specific substitutions at known locations.
Another strategy involves detection of mutations in duplex DNA containing single base mismatches (Shenk et al., 1975 Proc. Natl. Acad. Sci. USA 72:989). In this method some DNA:DNA mismatches are cleaved, to a limited extent, with the single strand specific nuclease Sl.